The nucleolus contains scores of small nucleolar RNA-protein complexes (snoRNPs), which modify nucleotides in rRNA (most snoRNPs do this) or participate in processing (cleavage) of prerRNA. The modifying snoRNPs form 2'-O-methylated nucleotides and pseudouridine, mostly in functionally important regions of the ribosome. The effects of these alterations are virtually unknown. The main unanswered question with the processing snoRNPs is their actual role. The proposal has two main aims -designed to answer these important questions. Both build on results from the previous grant. First, we showed that systematic depletion of nucleotide modifications from the reaction center region of the ribosome causes defects in translation. Second, we have affinity-isolated a conserved snoRNP (U14) required for processing and methylation of 18S rRNA, and identified several novel proteins, including factors already tied to ribosome synthesis. The major aims of our application are: 1) To determine the influence of nucleotide modifications in the mRNA decoding center and two other domains that interact dynamically with tRNA in the decoding region, and; 2) To determine the roles of the U14 snoRNP proteins in rRNA processing and modification. Effects of nucleotide modification will be investigated by depleting selected modifications from the target regions and examining ribosome synthesis and function. Studies of the U14 snoRNP will focus initially on defining the roles of the new snoRNP components in ribosome synthesis, by genetic depletion, mutation and affinity isolation strategies. Other goals include describing the assembly, structure and activity of the snoRNP complex. Results from the studies will yield valuable new insights into ribosome synthesis and function, and could shed light on the basis of established links between snoRNPs, aging and cancer.